Fusarium circinatum Nirenberg & O'Donnell, the pitch canker fungus, has been recently reported in Uruguay affecting Pinus taeda L. seedlings. The spread of this pathogen to plantations constitute a risk to forestry production. The aim of this work was to evaluate the inhibitory effect of live bacteria and their thermostable metabolites on F. circinatum growth in vitro. Four Bacillus subtilis strains and one of Burkholderia sp. isolated as P. taeda endophytes were evaluated as biological control agents of F. circinatum. Dual cultures between live bacteria and pathogen were performed. Furthermore, bacteria metabolites obtained from liquid cultures were sterilized and added to the culture media where fungus was grown. In this study all bacteria showed an antagonist effect on the pathogen growth arresting the mycelia at one cm of the edge of the bacteria colony. Bacteria thermostable metabolites reduced over 50% fungal growth. These results demonstrates that endophytic bacteria, well adapted to live in host tissues, constitute a good alternative to control F. circinatum affecting Pinus seedlings.

The pitch canker fungus Fusarium circinatum Nirenberg and O'Donnell is a destructive pathogen that affects several Pinus species (Barnard and Blakeslee, 1980; Viljoen et al., 1994). The symptom more frequently associated to this pathogen is the presence of large resinous cankers on the main trunk and lateral branches of trees but it can also be associated to roots, shoots, cones, and seedlings. In plant seedlings aerial symptoms do not appear until the pathogen reach the trunk from lesions at soil level resulting in plant discoloration and needles drying. The disease has been detected in south eastern USA (Kuhlman et al., 1982), Mexico, South Africa, Chile, Japan, and Spain (Kobayashi and Muramoto, 1989; Guerra-Santos, 1999; Wingfield et al., 2002; Perez Sierra et al., 2007). Recently, in Uruguay this pathogen was detected on Pinus taeda L. seedlings from nurseries mainly affecting stem collar (Alonso and Bettucci, 2009).

According to Cook and Baker (1983) biological control can be defined as a reduction of the amount of inoculum or disease produced by the activity of a pathogen, based on the use of natural enemies or the use of compounds derived from its metabolism. Then, the biological control offers an alternative to the chemical products, contributing to minimize the negative consequences for human health and environment (Kim et al., 2003). Fungal diseases are very frequent in nurseries and the chemical control of pathogens is the most common practice.

Bacillus subtilis has been identified as a potent antagonist against several fungal pathogens due to the production of antifungal compounds, antibiotics and proteases, hence is extensively used in agricultural systems (Todorova and Kozhuharova, 2009; Chen et al., 2009; Kinsella et al., 2009).

Burkholderia sp. is known to have beneficial effect on plant growth through the production of antifungal and other compounds that are able to suppress many soil-borne plant pathogens (Holmes et al., 1998). Burkholderia cepacia is an ubiquitous soil organism that can be easily obtained and it has been studied as biocontrol agent of plant disease (Leisinger and Margraff, 1979). Many of its metabolites have been isolated and identified thus verifying its inhibitory effect on different plant pathogens such as fungus, bacteria and yeasts (Sopheareth et al., 2006), particularly on species of Pythium, Botrytis, Fusarium, and Rhizotocnia (Sijam and Dikin, 2005; Quan et al., 2006).

The abuse and misuse of chemical products can cause environmental and human health-related risks. On the other hand, little work has been performed on biological control of forest pathogens. Identification and action mode of antifungal compounds produced by an antagonist need to be studied.

The aim of this work was to evaluate the antagonist effect of both live bacteria and their thermostable metabolites of four Bacillus subtilis strains and one of Burkholderia sp. on Fusarium circinatum growth.

MATERIALS AND METHODS

Fungal and bacteria isolatesFusarium circinatum strains used in this work were all isolated from symptomatic Pinus taeda seedlings from two Pinus nurseries from Rivera and Florida Departments in Uruguay. The fungal isolates were identified by macro and micromorphological characteristics and verified by molecular analysis using CIRC1A and CIRC4A specific primers for F. circinatum (Schweigkofler et al., 2004). The isolates were maintained in potato dextrose agar (PDA). Bacteria were present as endophytes from Pinus seedlings and were isolated from stem healthy tissues. Those showing inhibitory effect on fungal growth were selected. The identification of bacteria strains were performed by molecular analysis of 16S RNA region. The cultures were maintained on triptone soy agar (TSA).

Bacteria antagonist on F. circinatum growthTo evaluate the antagonist effect of different live bacteria, mycelia plugs from the edges of actively growing fungal cultures were placed in the center of Petri dish containing PDA. Four bacteria isolates were streaked on the same plates at equal distance from the fungal inocula. Plates with the fungal plug without bacteria were used as control. Plates were incubated at 25 °C for 5 d to evaluate the inhibition activity of bacteria on the fungus. Each treatment was replicated five times. The fungal strains used were Fc 2052, Fc2053, Fc2054, and Fc2057. Bacteria strains of Bacillus subtilis used were B1, B2, B3, B4, and one strain of Burkholderia sp. (B5).

Observations of mycelia of the interaction zone between fungi and bacteria were performed under microscope.

The activity of bacteria thermostable metabolites was also evaluated. Liquid cultures of bacteria were performed transferring colonies of each bacterium to a 250 mL Erlenmeyer flask containing 100 mL of potato dextrose broth (PDB) and then incubated in a rotary shaker at 27 °C and 180 rpm during 7 d. Ten milliliters of each flask were transferred to a new flask with 90 mL of PDA. These new flasks were sterilized during 16 min at 121 °C and 1 atm. The culture medium plus the metabolites were homogenized and 20 mL were placed on Petri dishes of 9 cm of diameter. Once the medium was solidified a plug of each fungal strain was placed in the centre of a dish. A fungal plug placed on PDA was used for control. Each treatment and control was replicated three times. Both treatments and controls were incubated at 25 °C during 9 d. After incubation for 120 h the diameter of the colonies was measured daily during 4 d and compared with controls. The measures were made from the centre of the fungal plug to the edge of the colony. Two measures were taken which were then averaged. Percentage of inhibition growth and the rate of growth were calculated. To determine if there were differences in the rate of growth between the four fungal strains tested a One Way ANOVA test was made using the Sigma Stat 3.5 program.

RESULTS AND DISCUSSION

Isolation of bacteria and screening of antifungal effect The screening of bacteria for antifungal activity against the Pinus pathogen F. circinatum showed that all of them exhibited growth inhibition against the pathogen. All the strains arrested the mycelium growth at 1 cm or more of the fungal colony margin (Figure 1). The development of an inhibition halo was observed between the fungal colonies and the bacteria inocula. This may be due to the production of bacterial metabolites that may diffuse in the culture medium and suppress the growth of F. circinatum. These results are consistent with those obtained by Nourozian et al. (2006) who evaluated the antagonist activity of different bacteria (Bacillus, Pseudomonas) against F. graminearum. They observed, in dual culture experiments, the formation of inhibition zones between bacteria and fungus.

The micromorphology of mycelia in the interaction zone showed a change in hyphal mode development, exhibiting empty, vacuolated and swollen hypha and a different ramification pattern.

Effect of thermostable metabolitesDespite metabolites of all strains showed an inhibitory effect against the fungus strains tested (Figure 2) all of them had a different incidence on the fungal growth (Figure 3).

There were significant differences among the bacterial strains. Growth inhibition on the strain Fc2052 was greater than to the other strains (p < 0.05) (Figure 2). On the other hand, from all strains of B. subtilis the strain B2 showed the lowest effect on the pathogen growth. Although the fungal strain Fc2053 showed a lesser growth than the control, the difference was not significant. The effect of metabolites of B. subtilis B1 on Fc2054 was greater than to other fungal strains. Metabolites from Burkholderia sp. evidenced a lesser effect on the growth of this pathogen. Metabolites of B1, B3, B4, and B5 reduced the growth of F. circinatum Fc2057over 50% (Figure 2).

These results showed that the metabolites of Bacillus and Burkholderia tested, reduced the rate of growth of F. circinatum although some differences among fungal strains were observed. These findings suggest the possibility of using B. subtilis as biocontrol agent of Pinus pathogen F. circinatum, consistently with other studies where Bacillus has inhibitory effect against Fusarium spp. and other plant pathogen fungi (Moita et al., 2005; Kinsella et al., 2009). Recently, Burkholderia spp. have been used as biocontrol agents against fungal disease, including Fusarium spp. (Quan et al., 2006).

CONCLUSIONS

The bacteria that were present as endophytes of Pinus taeda seedlings were symptomless colonizers and apparently adapted to host tissues. This can constitute an advantage for using them as biocontrol agent of the pitch canker fungus on this host. The biological control could be an alternative to reduce the incidence of the pathogen in nurseries in order to avoid the expansion of the disease to the field. The active thermostable metabolites are also a very interesting alternative to chemical control and to avoid the use of living organisms. Both, B. subtilis and Burkholderia are not frequently used in forest management.

ACKNOWLEDGEMENTS

We thank Estella Reginensi and Ana Clara Bianchi for the identification of the bacteria used in this study.